The major focus of our research is understanding the signals which regulate B lymphocyte activation. We have a special interest in signals delivered to B cells through their interactions with T lymphocytes, and in recent years have particularly focused upon B cell signaling via CD40. Our recent findings have impressed upon us the potential importance of physical interactions between CD40 and cytoplasmic signaling proteins in cholesterol-rich membrane microdomains, or rafts. It was found the engagement of CD40 on B lymphocytes leads to the rapid recruitment to the CD40 cytoplasmic domain of tumor necrosis factor associated factors (TRAFs) 2 and 3. Both CD40 and TRAFs 2 and 3 become associated with detergent-insoluble, cholesterol-enriched membrane microdomains, or rafts, and the raft-recruited TRAFs become posttranslationally modified, with the appearance of multiple higher MW forms. This is followed by initiation of proteasome-dependent degradation of these two TRAFs. The central hypothesis underlying the proposed studies is that physical compartmentalization in the plasma membrane serves to regulate the interactions between CD40 and the TRAFs, to orchestrate CD40 signaling pathways. It is also proposed that effects on membrane compartmentalization play an important role in interactions between receptors and signaling proteins, as well as in interactions between individual receptors, determining the ultimate outcome of the message delivered to the B cell. Our specific experimental goals are (1) To determine how interactions between CD40 and TRAFs 2 and 3 in membrane microdomains regulate CD40 signaling, by investigating the nature of the CD40-induced modification of raft-recruited TRAF2 and TRAF3, how this modification is regulated and what role it plays in CD40 signaling, and identifying the protein-protein associations mediating initiation of the CD40 signaling cascade in membrane microdomains; (2) To investigate the nature of the interactions between CD40 and TRAFs 1 and 6 which are quite distinct from those between CD40 and TRAFs 2 and 3, and determining their roles in CD40 effector functions; and (3) To investigate how physical compartmentalization in the plasma membrane regulates the interaction between signaling by CD40, the B cell antigen receptor, and Fas.